> In order to obtain the 5' sequence, I've come up with three approaches
> and would appreciate your comments/suggestions.
> 1) Rescreen the cDNA library using a DNA probe prepared from one of the
> nearly full size clones on hand. I'm trying this currently, but so far
> have only pulled out one clone that I have not yet sequenced.
> 2) Use a 5' fragment of the largest cDNA clone to sequence directly off
> transcripts extracted from tissue. The protein is fairly abundant when
> induced (app. 1%); should there be enough transcript to prepare cDNA for
> sequencing?
> 3) Use PCR-based RACE or SLIC to amplify the 5' end from either the cDNA
> library or transcripts. I've gotten varying estimates of the success rate
> of these methods. That fact, combined with the price of the kits (app.
> $400 from Gibco/BRL or Clontech) urges me to solicit advice. Do these
> approaches work? Is there a cheaper way to do this by buying individual
> enzymes and reagents?
The problem regarding RT/5' underrepresentation is serious only with cDNA
libraries that are oligo-dT primed, ie the cDNAs are initiated at the
extreme 3' end of the mRNA. A random primed cDNA library should have 5'
ends represented as well as 3'. You could screen a random primed cDNA
library with 5' portions of your sequence and should be able to get more 5'
information.
--
Fred Boyd, Ph.D. Voice- 612-624-8150
Lab. Med. Path. Fax- 612-616-2444
Cell Biol. Neuro. Lab- 612-624-8154
Box 609, UMHC
University of Minnesota E-Mail fredboyd at bmec.micro.umn.edu
Minneapolis, MN 55455